EP1915626B1 - Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues - Google Patents

Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues Download PDF

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EP1915626B1
EP1915626B1 EP06813477A EP06813477A EP1915626B1 EP 1915626 B1 EP1915626 B1 EP 1915626B1 EP 06813477 A EP06813477 A EP 06813477A EP 06813477 A EP06813477 A EP 06813477A EP 1915626 B1 EP1915626 B1 EP 1915626B1
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Prior art keywords
antibody
galnac
tissue
expression
cancer
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German (de)
English (en)
French (fr)
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EP1915626A2 (en
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Klaus W. Wagner
Avi J. Ashkenazi
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Genentech Inc
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Genentech Inc
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Definitions

  • the inventions described herein relate to methods and assays to detect biomarkers predictive of sensitivity of mammalian cells to death receptor agonist antibodies. More particularly, the inventions herein relate to methods and assays which detect molecules associated with the GalNac-T family of proteins which are predictive of sensitivity of mammalian cancer cells to DR4 or DR5 agonist antibodies.
  • TNF tumor necrosis factor
  • TNF-alpha tumor necrosis factor-alpha
  • TNF-beta tumor necrosis factor-beta
  • LT-beta lymphotoxin-beta
  • CD30 ligand CD27 ligand
  • CD40 ligand OX-40 ligand
  • 4-1BB ligand LIGHT
  • Apo-1 ligand also referred to as Fas ligand or CD95 ligand
  • Apo-2 ligand also referred to as Apo2L or TRAIL
  • Apo-3 ligand also referred to as TWEAK
  • APRIL OPG ligand
  • OPG ligand also referred to as RANK ligand, ODF, or TRANCE
  • TALL-1 also referred to as BlyS, BAFF or THANK
  • TNF receptor family members share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions, while others are found naturally as soluble proteins lacking a transmembrane and intracellular domain.
  • the extracellular portion of typical TNFRs contains a repetitive amino acid sequence pattern of multiple cysteine-rich domains (CRDs), starting from the NH 2 -terminus.
  • the ligand referred to as Apo-2L or TRAIL was identified several years ago as a member of the TNF family of cytokines. (see, e.g., Wiley et al., Immunity, 3:673-682 (1995 ); Pitti et al., J. Biol. Chem., 271:12697-12690 (1996 ); WO 97/01633 ; WO 97/25428 ; US Patent 5,763,223 issued June 9, 1998 ; US Patent 6,284,236 issued September 4, 2001 ).
  • the full-length native sequence human Apo2L/TRAIL polypeptide is a 281 amino acid long, Type II transmembrane protein.
  • Some cells can produce a natural soluble form of the polypeptide, through enzymatic cleavage of the polypeptide's extracellular region ( Mariani et al., J. Cell. Biol., 137:221-229 (1997 )). Crystallographic studies of soluble forms of Apo2L/TRAIL reveal a homotrimeric structure similar to the structures of TNF and other related proteins ( Hymowitz et al., Molec.
  • Apo2L/TRAIL unlike other TNF family members however, was found to have a unique structural feature in that three cysteine residues (at position 230 of each subunit in the homotrimer) together coordinate a zinc atom, and that the zinc binding is important for trimer stability and biological activity. (Hymowitz et al., supra; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000 )).
  • Apo2L/TRAIL may play a role in immune system modulation, including autoimmune diseases such as rheumatoid arthritis [see, e.g., Thomas et al., J. Immunol., 161:2195-2200 (1998 ); Johnsen et al., Cytokine, 11:664-672 (1999 ); Griffith et al., J. Exp. Med., 189:1343-1353 (1999 ); Song et al., J. Exp. Med., 191:1095-1103 (2000 )].
  • autoimmune diseases such as rheumatoid arthritis
  • Soluble forms of Apo2L/TRAIL have also been reported to induce apoptosis in a variety of cancer cells, including colon, lung, breast, prostate, bladder, kidney, ovarian and brain tumors, as well as melanoma, leukemia, and multiple myeloma (see, e.g., Wiley et al., supra; Pitti et al., supra; US Patent 6,030,945 issued February 29, 2000 ; US Patent 6,746,668 issued June 8, 2004 ; Rieger et al., FEBS Letters, 427:124-128 (1998 ); Ashkenazi et al., J. Clin.
  • Apo2L/TRAIL preparations may vary in terms of biochemical properties and biological activities on diseased versus normal cells, depending, for example, on the presence or absence of a tag molecule, zinc content, and % trimer content (See, Lawrence et al., Nature Med., Letter to the Editor, 7:383-385 (2001 ); Qin et al., Nature Med., Letter to the Editor, 7:385-386 (2001 )).
  • Apo2L/TRAIL has been found to bind at least five different receptors. At least two of the receptors which bind Apo2L/TRAIL contain a functional, cytoplasmic death domain.
  • One such receptor has been referred to as "DR4" (and alternatively as TR4 or TRAIL-R1) ( Pan et al., Science, 276:111-113 (1997 ); see also WO98/32856 published July 30, 1998 ; WO99/37684 published July 29, 1999 ; WO 00/73349 published December 7, 2000 ; US 2003/0036168 published February 20, 2003 ; US 6,433,147 issued August 13, 2002 ; US 6,461,823 issued October 8, 2002 , and US 6,342,383 issued January 29, 2002 ).
  • DR5 is reported to contain a cytoplasmic death domain and be capable of signaling apoptosis upon ligand binding (or upon binding a molecule, such as an agonist antibody, which mimics the activity of the ligand).
  • the crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et al., Molecular Cell, 4:563-571 (1999 ).
  • both DR4 and DR5 can trigger apoptosis independently by recruiting and activating the apoptosis initiator, caspase-8, through the death-domain-containing adaptor molecule referred to as FADD/Mort1 [ Kischkel et al., Immunity, 12:611-620 (2000 ); Sprick et al., Immunity, 12:599-609 (2000 ); Bodmer et al., Nature Cell Biol., 2:241-243 (2000 )].
  • DcR1, DcR2 and OPG which believed to function as inhibitors, rather than transducers of signaling
  • DCR1 also referred to as TRID, LIT or TRAIL-R3
  • TRID TRID, LIT or TRAIL-R3
  • anti-DR4 antibodies directed to the DR4 receptor and having agonistic or apoptotic activity in certain mammalian cells are described in, e.g., WO 99/37684 published July 29, 1999 ; WO 00/73349 published July 12, 2000 ; WO 03/066661 published August 14, 2003 . See, also, e.g., Griffith et al., J. Immunol., 162:2597-2605 (1999 ); Chuntharapai et al., J.
  • the invention disclosed herein provides methods and assays examining expression of one or more biomarkers in a mammalian tissue or cell sample, wherein the expression of one or more such biomarkers is predictive of whether the tissue or cell sample will be sensitive to agents such as anti-DR5 agonist antibodies.
  • the methods and assays examine expression of molecules in the GalNac-T family of proteins, in particular. GalNAc-T14 (see Wang H. et al 300, 738-744)
  • Apo2L/TRAIL As discussed above, most normal human cell types appear to be resistant to apoptosis induction by certain recombinant forms of Apo2L/TRAIL (Ashkenazi et al., supra; Walzcak et al., supra). It has also been observed that some populations of diseased human cell types (such as certain populations of cancer cells) are resistant to apoptosis induction by certain recombinant forms of Apo2L/TRAIL ( Ashkenazi et al., J. Clin. Invest., 1999 , supra ; Walczak et al., Nature Med., 1999 , supra ).
  • information obtained from an assay to detect GalNac-T14 expression in a mammalian tissue or cell sample can provide physicians with useful data that can be used to determine an optimal therapeutic regimen (using death receptor agonist antibodies) for patients suffering from a disorder such as cancer.
  • the invention provides methods for predicting the sensitivity of a mammalian cancer tissue sample or cell sample to a death receptor antibody, the method comprising examining the tissue sample or cell sample to detect expression of GalNac-T14, wherein expression of said GalNac-T14 is predictive that said tissue sample or cell sample is sensitive to apoptosis-inducing activity of the death receptor antibody, wherein said death receptor antibody comprises a DR4 agonist antibody or a DR5 agonist antibody which is capable of inducing apoptosis.
  • the methods may be conducted in a variety of assay formats, including assays detecting mRNA and/or protein expression, enzymatic activity assays and others discussed herein.
  • the present invention provides the use of a death receptor antibody in the manufacture of a medicament for the treatment of cancer in a mammalian subject, wherein a tissue sample or cell sample from the subject has been determined to express GalNac-T14 and expression of GalNac-T14 is predictive that said tissue sample or cell sample is sensitive to apoptosis-inducing activity of the death receptor antibody, and wherein the death receptor antibody comprises a DR4 agonist antibody or a DR5 agonist antibody capable of inducing apoptosis.
  • the present invention provides a death receptor antibody for use in a method of treating cancer in a mammalian subject, wherein a tissue sample or cell sample from the subject has been determined to express GalNac-T14 and expression of GalNac-T14 is predictive that said tissue sample or cell sample is sensitive to apoptosis-inducing activity of the death receptor antibody, and wherein the death receptor antibody comprises a DR4 agonist antibody or a DR5 agonist antibody capable of inducing apoptosis.
  • examination of the expression of one or more biomarkers may be conducted in a variety of assay formats, including assays detecting mRNA and/or protein expression, enzymatic activity, and others discussed herein.
  • the methods also comprise examining the tissue or cell sample for expression of DR4, DR5, DcR1, or DcR2 receptors.
  • the methods comprise treating cancer in a mammal.
  • the methods comprise, in addition to the administration of an effective amount of death receptor agonist antibody, the administration of chemotherapeutic agent(s) or radiation therapy to said mammal.
  • Apo2L/TRAIL refers to a polypeptide sequence which includes amino acid residues 114-281, inclusive, 95-281, inclusive, residues 92-281, inclusive, residues 91-281, inclusive, residues 41-281, inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, of the amino acid sequence shown in Figure 1 , as well as biologically active fragments, deletional, insertional, or substitutional variants of the above sequences.
  • the polypeptide sequence comprises residues 114-281 of Figure 1 , and optionally, consists of residues 114-281 of Figure 1 .
  • substitutional variants include those identified, for example, as “D203A”; “D218A” and “D269A.” This nomenclature is used to identify Apo2L/TRAIL variants wherein the aspartic acid residues at positions 203, 218, and/or 269 (using the numbering shown in Figure 1 ) are substituted by alanine residues.
  • the Apo2L variants may comprise one or more of the alanine substitutions which are recited in Table I of published PCT application WO 01/00832 .
  • substitutional variants include one or more of the residue substitutions identified in Table I of WO 01/00832 published January 4, 2001 .
  • the definition also encompasses a native sequence Apo2L/TRAIL isolated from an Apo2L/TRAIL source or prepared by recombinant or synthetic methods.
  • the Apo2L/TRAIL of the invention includes the polypeptides referred to as Apo2L/TRAIL or TRAIL disclosed in PCT Publication Nos. WO97/01633 and WO97/25428 .
  • the terms "Apo2L/TRAIL” or “Apo2L” are used to refer generally to forms of the Apo2L/TRAIL which include monomer, dimer or trimer forms of the polypeptide. All numbering of amino acid residues referred to in the Apo2L sequence use the numbering according to Figure 1 , unless specifically stated otherwise. For instance, "D203" or “Asp203” refers to the aspartic acid residue at position 203 in the sequence provided in Figure 1 .
  • the ECD will consist of a soluble, extracellular domain sequence of the polypeptide which is free of the transmembrane and cytoplasmic or intracellular domains (and is not membrane bound).
  • Particular extracellular domain sequences of Apo-2L/TRAIL are described in PCT Publication Nos. WO97/01633 and WO97/25428 .
  • Apo2L/TRAIL monomer or "Apo2L monomer” refers to a covalent chain of an extracellular domain sequence of Apo2L.
  • Apo2L/TRAIL dimer or "Apo2L dimer” refers to two Apo-2L monomers joined in a covalent linkage via a disulfide bond.
  • the term as used herein includes free standing Apo2L dimers and Apo2L dimers that are within trimeric forms of Apo2L (i.e., associated with another, third Apo2L monomer).
  • Apo2L/TRAIL trimer or "Apo2L trimer” refers to three Apo2L monomers that are non-covalently associated.
  • Apo2L/TRAIL aggregate is used to refer to self-associated higher oligomeric forms of Apo2L/TRAIL, such as Apo2L/TRAIL trimers, which form, for instance, hexameric and nanomeric forms of Apo2L/TRAIL. Determination of the presence and quantity of Apo2L/TRAIL monomer, dimer, or trimer (or other aggregates) may be made using methods and assays known in the art (and using commercially available materials), such as native size exclusion HPLC (“SEC”), denaturing size exclusion using sodium dodecyl sulphate (“SDS-SEC”), reverse phase HPLC and capillary electrophoresis.
  • SEC native size exclusion HPLC
  • SDS-SEC denaturing size exclusion using sodium dodecyl sulphate
  • reverse phase HPLC capillary electrophoresis.
  • Apo-2 ligand receptor includes the receptors referred to in the art as "DR4" and "DR5" whose polynucleotide and polypeptide sequences are shown in Figures 2 and 3 respectively.
  • Pan et al. have described the TNF receptor family member referred to as "DR4" ( Pan et al., Science, 276:111-113 (1997 ); see also WO98/32856 published July 30, 1998 ; WO 99/37684 published July'29, 1999 ; WO 00/73349 published December 7, 2000 ; US 6,433,147 issued August 13, 2002 ; US 6,461,823 issued October 8, 2002 , and US 6,342,383 issued January 29, 2002 ).
  • a native sequence Apo-2L receptor can have the amino acid sequence of naturally-occurring Apo-2L receptor from any mammal.
  • Such native sequence Apo-2L receptor can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence Apo-2L receptor" specifically encompasses naturally-occurring truncated or secreted forms of the receptor (e.g., a soluble form containing, for instance, an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants.
  • Receptor variants may include fragments or deletion mutants of the native sequence Apo-2L receptor.
  • DR5 receptor antibody “DR5 antibody”, or “anti-DR5 antibody” is used in a broad sense to refer to antibodies that bind to at least one form of a DR5 receptor, such as the 1-411 sequence shown in Figures 3A or the 1-440 sequence shown in Figures 3B-3C , or extracellular domain thereof.
  • the DR5 antibody is fused or linked to a heterologous sequence or molecule.
  • the heterologous sequence allows or assists the antibody to form higher order or oligomeric complexes.
  • the DR5 antibody binds to DR5 receptor but does not bind or cross-react with any additional Apo-2L receptor (e.g. DR4, DcR1, or DcR2).
  • the antibody is an agonist of DR5 signalling activity.
  • DR4 receptor antibody “DR4 antibody”, or “anti-DR4 antibody” is used in a broad sense to refer to antibodies that bind to at least one form of a DR4 receptor or extracellular domain thereof.
  • the DR4 antibody is fused or linked to a heterologous sequence or molecule.
  • the heterologous sequence allows or assists the antibody to form higher order or oligomeric complexes.
  • the DR4 antibody binds to DR4 receptor but does not bind or cross-react with any additional Apo-2L receptor (e.g. DR5, DcR1, or DcR2).
  • the antibody is an agonist of DR4 signalling activity.
  • the DR4 antibody of the invention binds to a DR4 receptor at a concentration range of about 0.1 nM to about 20 mM as measured in a BIAcore binding assay.
  • the DR4 antibodies of the invention exhibit an Ic 50 value of about 0.6 nM to about 18 mM as measured in a BIAcore binding assay.
  • UDP-N-.acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T14", "pp-GalNac-T14", “GalNac-T14”, “GALNT14” are used herein to refer a type II membrane protein having characteristic features of the GalNac-T family of molecules comprising a N-terminal cytoplasmic domain, transmembrane domain, stem region and catalytic domain.
  • the human GalNac-T14 molecule contains 1659 base pairs encoding a 552 amino acid protein, as shown in Figure 4A .
  • the full length human cDNA has been deposited in GenBank as Accession No.
  • subject or “patient” is meant any single subject for which therapy is desired, including humans. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
  • tissue or cell sample is meant a collection of similar cells obtained from a tissue of a subject or patient.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may also be primary or cultured cells or cell lines.
  • the tissue or cell sample is obtained from a primary or metastatic tumor.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • a "section" of a tissue sample is meant a single part or piece of a tissue sample, e . g . a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention, provided that it is understood that the present invention comprises a method whereby the same section of tissue sample is analyzed at both morphological and molecular levels, or is analyzed with respect to both protein and nucleic acid.
  • gene any nucleic acid sequence or portion thereof with a functional role in encoding or transcribing a protein or regulating other gene expression.
  • the gene may consist of all the nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein.
  • the nucleic acid sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences or unique adjacent regions to the gene.
  • label when used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable or complementary determining regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the [ ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a, few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097 ; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993 ).
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al.; Nature, 352:624-628 (1991 ) and Marks et al., J. Mol. Biol., 222:581-597 (1991 ), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass; as well as fragments of such antibodies, so long as they exhibit the desired biological activity ( U.S. Patent No. 4,816,567 ; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984 )).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequence
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g . old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences ( US Pat No. 5,693,780 ).
  • a non-human primate e.g . old World Monkey, such as baboon, rhesus or cynomolgus monkey
  • human constant region sequences US Pat No. 5,693,780
  • Humanized forms of non-human (e.g ., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )) and/or those residues from a "hypervariable loop" (e.g.
  • An antibody "which binds" an antigen of interest is one capable of binding that antigen with sufficient affinity and/or avidity such that the antibody is useful as a therapeutic or diagnostic agent for targeting a cell expressing the antigen.
  • immunotherapy will refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody may be an unconjugated or “naked” antibody, or the antibody may be conjugated or fused with heterologous molecule(s) or agent(s), such as one or more cytotoxic agent(s), thereby generating an "immunoconjugate".
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • an agent e.g. Apo2L/TRAIL, anti-DR4 or DR5 antibody etc.
  • an agent e.g. Apo2L/TRAIL, anti-DR4 or DR5 antibody etc.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g ., I 131 , I 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN TM ); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycin
  • calicheamicin especially calicheamicin gamma1I and calicheamicin phiI1, see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994 ); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin TM ) (
  • paclitaxel TAXOL°, Bristol-Myers Squibb Oncology, Princeton, NJ
  • doxetaxel TAXOTERE°, Rhône-Poulenc Rorer, Antony, France
  • chlorambucil gemcitabine (Gemzar TM ) 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine TM ); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including Nolvadex TM
  • raloxifene including Nolvadex TM
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LY117018 onapristone
  • toremifene Fraston TM
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace TM ) , exemestane, formestane, fadrozole, vorozole (Rivisor TM ), letrozole (Femara TM ), and anastrozole (Arimidex TM ); and anti-androgens such
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Those agents that arrest G1 also spill over into S-phase arrest, for, example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13 .
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • apoptosis and apoptotic activity are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that is typically accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function.
  • This activity can be determined and measured, for instance, by cell viability assays (such as Alamar blue assays or MTT assays), FACS analysis, caspase activation, DNA fragmentation (see, for example, Nicoletti et al., J. Immunol. Methods, 139:271-279 (1991 ), and poly-ADP ribose polymerase, "PARP", cleavage assays known in the art.
  • disorders in general refers to any condition that would benefit from treatment with the compositions described herein, including any diseases or disorder that can be treated by effective amounts of Apo2L/TRAIL, an anti-DR4 antibody, and/or an anti-DR5 antibody.
  • This includes chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in question.
  • disorders to be treated herein include benign and malignant cancers; inflammatory, angiogenic, and immunologic disorders, autoimmune disorders, arthritis (including rheumatoid arthritis), multiple sclerosis, and HIV/AIDS.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma.
  • cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, non-hodgkin's lymphoma, gastrointestinal (tract), cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer.
  • tagged when used herein refers to a chimeric molecule comprising an antibody or polypeptide fused to a "tag polypeptide".
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made or to provide some other function, such as the ability to oligomerize (e.g. as occurs with peptides having leucine zipper domains), yet is short enough such that it generally does not interfere with activity of the antibody or polypeptide.
  • the tag polypeptide preferably also is fairly unique so that a tag-specific antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 to about 50 amino acid residues (preferably, between about 10 to about 20 residues).
  • divalent metal ion refers to a metal ion having two positive charges.
  • divalent metal ions include but are not limited to zinc, cobalt, nickel, cadmium, magnesium, and manganese.
  • Particular forms of such metals that may be employed include salt forms (e.g., pharmaceutically acceptable salt forms), such as chloride, acetate, carbonate, citrate and sulfate forms of the above mentioned divalent metal ions.
  • a divalent metal ion for use in the present invention is zinc, and preferably, the salt form, zinc sulfate or zinc chloride.
  • Isolated when used to describe the various peptides or proteins disclosed herein, means peptide or protein that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the peptide or protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the peptide or protein will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain, or (3) to homogeneity by mass spectroscopic or peptide mapping techniques.
  • Isolated material includes peptide or protein in situ within recombinant cells, since at least one component of its natural environment will not be present. Ordinarily, however, isolated peptide or protein will be prepared by at least one purification step.
  • Percent (%) amino acid sequence identity with respect to the sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences being compared. For purposes herein, percent amino acid identity values can be obtained using the sequence comparison computer program, ALIGN-2, which was authored by Genentech, Inc.
  • ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, CA. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired identity between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995 ).
  • High stringency conditions are identified by those that: (1) employ low ionic strength and high temperature for washing; 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent; 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50'mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2
  • Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989 , and include overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • primer refers to oligonucleotide sequences that hybridize to a complementary RNA or DNA target polynucleotide and serve as the starting points for the stepwise synthesis of a polynucleotide from mononucleotides by the action of a nucleotidyltransferase, as occurs for example in a polymerase chain reaction.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ).
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US Patent No. 5,500,362 or 5,821,337.
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998 ).
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and carry out ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes monocytes
  • cytotoxic T cells and neutrophils cytotoxic T cells and neutrophils
  • Fc receptor or “FcR” are used to describe a receptor, that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the homozygous valine Fc ⁇ RIIIa Fc ⁇ RIIIa-158V has been shown to have a higher affinity for human IgGl and mediate increased ADCC in vitro relative to homozygous phenylalanine Fc ⁇ RIIIa Fc ⁇ RIIIa-158F) or heterozygous Fc ⁇ RIIIa-158F/V) receptors.
  • “Complement dependent cytotoxicity” or “CDC” refer to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule ( e . g . an antibody) complexed with a cognate antigen.
  • a CDC assay e.g . as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996 ), may be performed.
  • the methods and assays disclosed herein are directed to the examination of expression of one or more biomarkers in a mammalian tissue or cell sample, wherein the determination of that expression of one or more such biomarkers is predictive or indicative of whether the tissue or cell sample will be sensitive to agents such as Apo2L/TRAIL and/or death receptor antibodies such as anti-DR5 agonist antibodies or anti-DR4 agonist antibodies.
  • the methods and assays include those which examine expression of members of the GalNac-T family of molecules, including GalNac-T14 and GalNac-T3.
  • the disclosed methods and assays can provide for convenient, efficient, and potentially cost-effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients.
  • a patient having been diagnosed with cancer or an immune related condition could have a biopsy performed to obtain a tissue or cell sample, and the sample could be examined by way of various in vitro assays to determine whether the patient's cells would be sensitive to a therapeutic agent such as Apo2L/TRAIL or death receptor antibody.
  • the invention provides methods for predicting the sensitivity of a mammalian tissue or cell sample (such as a cancer cell) to Apo2L/TRAIL or a death receptor agonist antibody.
  • a mammalian tissue or cell sample is obtained and examined for expression of GalNac-T14.
  • the methods may be conducted in a variety of assay formats, including assays detecting mRNA expression, protein expression (such as immunohistochemistry assays) and biochemical assays detecting enzymatic UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase activity.
  • GalNac-T14 biomarkers in (or on) said tissues or cells will be predictive that such tissues or cells will be sensitive to the biological effects of Apo2L/TRAIL and/or death receptor antibody.
  • expression of various biomarkers such as GalNac-T14 in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including but not limited to, immunohistochemical and/or Western analysis, quantitative blood based assays (as for example Serum ELISA) (to examine, for example, levels of protein expression), biochemical enzymatic activity assays, in situ hybridization, Northern analysis and/or PCR analysis of mRNAs, and genomic Southern analysis (to examine, for example, gene deletion or amplification), as well as any one of the wide variety of assays that can be performed by gene and/or tissue array analysis.
  • immunohistochemical and/or Western analysis quantitative blood based assays (as for example Serum ELISA) (to examine, for example, levels of protein expression), biochemical enzymatic activity assays, in situ hybridization, Northern analysis and/or PCR analysis of mRNAs, and genomic Southern analysis (to examine, for example, gene deletion or amplification
  • Optional methods of the invention include protocols which examine or test for presence of GalNac-T14 in a mammalian tissue or cell sample.
  • a variety of methods for detecting GalNac-T14 can be employed and include, for example, immunohistochemical analysis, immunoprecipitation, Western blot analysis, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (FACS), and immunoprecipitation followed by MS, monosaccharide analysis.
  • an optional method of detecting the expression of GalNac-T14 in a tissue or sample comprises contacting the sample with an anti-GalNac-T14 antibody and then detecting the binding of the antibody to GalNac-T14 in the sample.
  • the expression of GalNac-T14 in a sample is examined using immunohistochemistry and staining protocols.
  • Immunohistochemical staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample.
  • Immunohistochemistry (“IHC") techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods.
  • tissue or cell sample from a mammal may be used.
  • samples include, but are not limited to, cancer cells such as colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemia cancer cells.
  • the samples include non-small cell lung cancer cells, pancreatic cancer cells or non-hodgkin's lymphoma cancer cells.
  • the sample can be obtained by a variety of procedures known in the art including, but not limited to surgical excision, aspiration or biopsy.
  • the tissue may be fresh or frozen.
  • the sample is fixed and embedded in paraffin or the like.
  • the tissue sample may be fixed ( i.e . preserved) by conventional methodology (See e . g ., " Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3rd edition (1960) Lee G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York ; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C .).
  • a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed.
  • the length of fixation depends upon the size of the tissue sample and the fixative used. By way of example, neutral buffered formalin, Bouin's or paraformaldehyde, may be used to fix a sample.
  • the sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, one may section the tissue and fix the sections obtained.
  • the tissue sample may be embedded and processed in paraffin by conventional methodology (See e.g., "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra).
  • paraffin that may be used include, but are not limited to, Paraplast, Broloid, and Tissuemay.
  • the sample may be sectioned by a microtome or the like (See e.g., "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra). By way of example for this procedure, sections may range from about three microns to about five microns in thickness.
  • the sections may be attached to slides by several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine and the like.
  • the paraffin embedded sections may be attached to positively charged slides and/or slides coated with poly-L-lysine.
  • the tissue sections are generally deparaffinized and rehydrated to water.
  • the tissue sections may be deparaffinized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols may be used (See e.g., " Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra ).
  • commercially available deparaffinizing non-organic agents such as Hemo-De7 (CMS, Houston, Texas) may be used.
  • a tissue section may be analyzed using IHC.
  • IHC may be performed in combination with additional techniques such as morphological staining and/or fluorescence in-situ hybridization.
  • Two general methods of IHC are available; direct and indirect assays.
  • binding of antibody to the target antigen e.g., GalNac-T14
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction.
  • a labeled primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety.
  • Numerous labels are available which can be generally grouped into the following categories:
  • enzyme-substrate combinations include, for example:
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody.
  • indirect conjugation of the label with the antibody can be achieved.
  • tissue section prior to, during or following IHC may be desired.
  • epitope retrieval methods such as heating the tissue sample in citrate buffer may be carried out (see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996 )).
  • the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample.
  • Appropriate conditions for achieving this can be determined by routine experimentation.
  • the extent of binding of antibody to the sample is determined by using any one of the detectable labels discussed above.
  • the label is an enzymatic label (e.g. HRPO) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3'-diaminobenzidine chromogen.
  • the enzymatic label is conjugated to antibody which binds specifically to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti-rabbit antibody).
  • the antibodies employed in the IHC analysis to detect expression of GalNac-T14 are anti-GalNac-Tl4 antibodies.
  • antibodies to other GalNac-T antigens which have cross-reactivity with GalNac-T14 may be employed.
  • the anti-GalNac-T14 antibody is a monoclonal antibody.
  • Staining intensity criteria may be evaluated as follows: TABLE 1 Staining Pattern Score No staining is observed in cells. 0 Faint/barely perceptible staining is detected in more than 10% of the cells. 1+ Weak to moderate staining is observed in more than 10% of the cells. 2+ Moderate to strong staining is observed in more than 10% of the cells. 3+
  • a staining pattern score of about 2+ or higher in such an IHC assay is believed to be predictive or indicative of sensitivity of a mammalian cell (such as a mammalian cancer cell) to Apo2L/TRAIL or a death receptor agonist antibody.
  • the sample may be contacted with an antibody specific for said biomarker under conditions sufficient for an antibody-biomarker complex to form; and then detecting said complex.
  • the presence of the biomarker may be accomplished in a number of ways, such as by Western blotting (with or without immunoprecipitation) and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum.
  • a wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043 , 4,424,279 and 4,018,653 . These include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays.
  • These assays also include direct binding of a labelled antibody to a target biomarker.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate, and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of biomarker.
  • a simultaneous assay in which both sample and labelled antibody are added simultaneously to the bound antibody.
  • a first antibody having specificity for the biomarker is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to 40°C such as between 25° C and 32° C inclusive) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the biomarker. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the molecular marker.
  • An alternative method involves immobilizing the target biomarkers in the sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase; glucose oxidase, - galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the first antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of biomarker which was present in the sample.
  • fluorescent compounds such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a Particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labelled antibody As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the molecular marker of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • Methods of the invention further include protocols which examine the presence and/or expression of GalNac-T14 mRNA in a tissue or cell sample.
  • Methods for the evaluation of mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled GalNac-T14 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for GalNac-T14, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • complementary DNA probes such as in situ hybridization using labeled GalNac-T14 riboprobes, Northern blot and related techniques
  • nucleic acid amplification assays such as RT-PCR using complementary primers specific for GalNac-T14, and other amplification type detection methods, such as, for example, branched DNA,
  • Tissue or cell samples from mammals can be conveniently assayed for, e.g., GalNac-T14 mRNAs using Northern, dot blot or PCR analysis.
  • RT-PCR assays such as quantitative PCR assays are well known in the art.
  • a method for detecting a GalNac-T14 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a GalNac-T14 polynucleotide as sense and antisense primers to amplify GalNac-T14 cDNAs therein; and detecting the presence of the amplified GalNac-T14 cDNA.
  • such methods can include one or more steps that allow one to determine the levels of GalNac-T14 mRNA in a biological sample (e.g. by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).
  • the sequence of the amplified GalNac-T14 cDNA can be determined.
  • Material embodiments of this aspect of the invention include GalNac-T14 primers and primer pairs, which allow the specific amplification of the polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
  • Probes may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • Such probes and primers can be used to detect the presence of GalNac-T14 polynucleotides in a sample and as a means for detecting a cell expressing GalNac-T14 proteins.
  • primers and probes may be prepared based on the sequences provided in herein and used effectively to amplify, clone and/or determine the presence and/or levels of GalNac-T14 mRNAs.
  • Optional methods of the invention include protocols which examine or detect mRNAs, such as GalNac-T14 mRNAs, in a tissue or cell sample by microarray technologies.
  • mRNAs such as GalNac-T14 mRNAs
  • test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes.
  • the probes are then hybridized to an array of nucleic acids immobilized on a solid support.
  • the array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes that have potential to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • Microarray technology utilizes nucleic acid hybridization techniques and computing technology to evaluate the mRNA expression profile of thousands of genes within a single experiment.
  • WO 01/75166 published October 11, 2001 ;
  • U.S. 5,700,637 , U.S. Patent 5,445,934 , and U.S. Patent 5,807,522 Lockart, Nature Biotechnology, 14:1675-1680 (1996 ); Cheung, V.G. et al., Nature Genetics 21(Suppl):15-19 (1999 ) for a discussion of array fabrication).
  • DNA microarrays are miniature arrays containing gene fragments that are either synthesized directly onto or spotted onto glass or other substrates. Thousands of genes are usually represented in a single array.
  • a typical microarray experiment involves the following steps: 1. preparation of fluorescently labeled target from RNA isolated from the sample, 2. hybridization of the labeled target to the microarray, 3. washing, staining, and scanning of the array, 4. analysis of the scanned image and 5. generation of gene expression profiles.
  • oligonucleotide usually 25 to 70 mers
  • gene expression arrays containing PCR products prepared from cDNAs In forming an array, oligonucleotides can be either prefabricated and spotted to the surface or directly synthesized on to the surface (in situ).
  • the Affymetrix GeneChip® system is a commerically available microarray system which comprises arrays fabricated by direct synthesis of oligonucleotides on a glass surface.
  • Probe/Gene Arrays Oligonucleotides, usually 25 mers, are directly synthesized onto a glass wafer by a combination of semiconductor-based photolithography and solid phase chemical synthesis technologies. Each array contains up to 400,000 different oligos and each oligo is present in millions of copies. Since oligonucleotide probes are synthesized in known locations on the array, the hybridization patterns and signal intensities can be interpreted in terms of gene identity and relative expression levels by the Affymetrix Microarray Suite software.
  • Each gene is represented on the array by a series of different oligonucleotide probes.
  • Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide.
  • the perfect match probe has a sequence exactly complimentary to the particular gene and thus measures the expression of the gene.
  • the mismatch probe differs from the perfect match probe by a single base substitution at the center base position, disturbing the binding of the target gene transcript. This helps to determine the background and nonspecific hybridization that contributes to the signal measured for the perfect match oligo.
  • the Microarray Suite software subtracts the hybridization intensities of the mismatch probes from those of the perfect match probes to determine the absolute or specific intensity value for each probe set.
  • Probes are chosen based on current information from Genbank and other nucleotide repositories. The sequences are believed to recognize unique regions of the 3' end of the gene.
  • a GeneChip Hybridization Oven (“rotisserie” oven) is used to carry out the hybridization of up to 64 arrays at one time.
  • the fluidics station performs washing and staining of the probe arrays. It is completely automated and contains four modules, with each module holding one probe array. Each module is controlled independently through Microarray Suite software using preprogrammed fluidics protocols.
  • the scanner is a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • the computer workstation with Microarray Suite software controls the fluidics station and the scanner.
  • Microarray Suite software can control up to eight fluidics stations using preprogrammed hybridization, wash, and stain protocols for the probe array.
  • the software also acquires and converts hybridization intensity data into a presence/absence call for each gene using appropriate algorithms.
  • the software detects changes in gene expression between experiments by comparison analysis and formats the output into .txt files, which can be used with other software programs for further data analysis.
  • Fluorescent in-situ hybridization (FISH) assay may also be used to detect expression of the biomarker mRNA using labeled probes.
  • FISH Fluorescent in-situ hybridization
  • the expression of a selected biomarker may also be assessed by examining gene deletion or gene amplification.
  • Gene deletion or amplification may be measured by any one of a wide variety of protocols known in the art, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA ( Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980 )), dot blotting (DNA analysis), or in situ hybridization (e.g., FISH), using an appropriately labeled probe, cytogenetic methods or comparative genomic hybridization (CGH) using an appropriately labeled probe.
  • these methods may be employed to detect deletion of amplification of GalNac-T14 genes.
  • methylation status of the biomarker such as GalNac-Tl4 gene
  • a tissue or cell sample Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes.
  • assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes which cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands.
  • MSP methylation specific PCR
  • MSP methylation specific PCR
  • This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995 ; De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999 ); Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536 ); and Lethe et al., Int. J. Cancer 76(6): 903-908 (1998 ).
  • GalNac-T14 expression of GalNac-T14 in a tissue or cell sample may also be examined by way of functional or activity-based assays. For instance, one may conduct assays known in the art to determine or detect the presence of the given enzymatic N-aetylgalactosaminyltransferase activity in the tissue or cell sample. (see, e.g., Bennett et al., J. Biol. Chem., 271:17006-17012 (1996 ); Wang et al., BBRC, 300:738-744 (2003 ); Hang et al., supra, available May 2005 at www.sciencedirect.com.
  • the tissue or cell sample may also be examined for the expression of Apo2L/TRAIL or receptors in the sample which bind death receptor antibody.
  • Apo2L/TRAIL binds to at least five different receptors: DR4, DR5, DcR1, DcR2, and OPG.
  • the expression of Apo2L/TRAIL, DR4, DR5, DcR1, DcR2 and/or OPG can be detected on the mRNA level and on the protein level.
  • the IHC techniques described above may be employed to detect the presence of one of more such molecules in the sample.
  • a tissue or sample in methods in which a tissue or sample is being examined not only for the presence of GalNac-T14 marker, but also for the presence, e.g., DR4, DR5 or DcR1, separate slides may be prepared from the same tissue or sample, and each slide tested with a reagent specific for each specific biomarker or receptor.
  • a single slide may be prepared from the tissue or cell sample, and antibodies directed to each biomarker or receptor may be used in connection with a multi-color staining protocol to allow visualization and detection of the respective biomarkers or receptors.
  • an effective amount of the death receptor antibody may be administered to the mammal to treat a disorder, such as cancer or immune related disorder which is afflicting the mammal.
  • a disorder such as cancer or immune related disorder which is afflicting the mammal.
  • Diagnosis in mammals of the various pathological conditions described herein can be made by the skilled practitioner. Diagnostic techniques are available in the art which allow, e.g., for the diagnosis or detection of cancer or immune related disease in a mammal. For instance, cancers may be identified through techniques, including but not limited to, palpation, blood analysis, x-ray, NMR and the like. Immune related diseases can also be readily identified.
  • the death receptor antibody can be administered in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • administration may be performed through mini-pump infusion using various commercially available devices.
  • Effective dosages and schedules for administering death receptor antibody may be determined empirically, and making such determinations is within the skill in the art. Single or multiple dosages may be employed. It is presently believed that an effective dosage or amount of Apo2L/TRAIL used alone may range from about 1 ⁇ g/kg to about 100 mg/kg of body weight or more per day. Interspecies scaling of dosages can be performed in a manner known in the art, e.g. , as disclosed in Mordenti et al., Pharmaceut. Res., 8:1351 (1991 ).
  • the one or more other therapies may include but are not limited to, administration of radiation therapy, cytokine(s), growth inhibitory agent(s), chemotherapeutic agent(s), cytotoxic agent(s), tyrosine kinase inhibitors, ras farnesyl transferase inhibitors, angiogenesis inhibitors, and cyclin-dependent kinase inhibitors which are known in the art and defined further with particularity above. It is contemplated that such other therapies may be employed as an agent separate from the death receptor antibody. In addition, therapies based on therapeutic antibodies that target tumor antigens such as Rituxan TM or Herceptin TM as well as anti-angiogenic antibodies such as anti-VEGF.
  • Preparation and dosing schedules for chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD. (1992 ). The chemotherapeutic agent may precede, or follow administration of the death receptor antibody, or may be given simultaneously therewith.
  • VEGF vascular endothelial factor
  • two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient.
  • treated cells in vitro can be analyzed. Where there has been in vivo treatment, a treated mammal can be monitored in various ways well known to the skilled practitioner. For instance, tumor cells can be examined pathologically to assay for necrosis or serum can be analyzed for immune system responses.
  • kits or articles of manufacture may be used.
  • Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a probe that is or can be detectably labeled.
  • probe may be an antibody or polynucleotide specific for GalNac-T14 protein or a GalNac-T14 gene or message, respectively.
  • the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
  • a reporter-means such as a biotin-binding protein, such as avidin or streptavidin
  • the kit typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters,. needles, syringes, and package inserts with instructions for use.
  • a label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • kits have a number of embodiments.
  • a typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a primary antibody that binds to a GalNac-T14 polypeptide sequence, the label on said container indicates that the composition can be used to evaluate the presence of GalNac-T14 proteins in at least one type of mammalian cell, and instructions for using the GalNac-T14 antibody for evaluating the presence of proteins in at least one type of mammalian cell.
  • the kit can further comprise a set of instructions and materials for preparing a tissue sample and applying antibody and probe to the same section of a tissue sample.
  • the kit may include both a primary and secondary antibody, wherein the secondary antibody is conjugated to a label, e.g. , an enzymatic label.
  • kits comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a polynucleotide that hybridizes to a complement of the GalNac-T14 polynucleotide under stringent conditions, the label on said container indicates that the composition can be used to evaluate the presence of GalNac-T14 in at least one type of mammalian cell, and instructions for using the GalNac-T14 polynucleotide for evaluating the presence of GalNac-T14 RNA or DNA in at least one type of mammalian cell.
  • kits include one or more buffers (e.g ., block buffer, wash buffer, substrate buffer, etc), other reagents such as substrate (e.g. , chromogen) which is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s) etc.
  • buffers e.g ., block buffer, wash buffer, substrate buffer, etc
  • substrate e.g. , chromogen
  • control samples positive and/or negative controls
  • Non-small cell lung cancer (NSCLC) lines H2122, A427, H647, SK-MES-1, H838, H358, H2126, H460, H1703, H2405, H650, H1568, H1666, H322T, SW1573, H292, H1650, H522, EKVX, H661, H23, LXFL 529, H226, A549, H1781, H1299, HOP 62, H2009, HOP 92, H1793, H1975, H1651, calu-1, H1435, HOP 18, H520,H441, H2030, H1155, H1838, H596, HLFa; Pancreatic cancer lines: Panc 05.04, BxPC3, HPAC, SU.86.86, HuP-T3, PSN1, Panc 08.13, MiaPaCa-2, PA-TU-8988T, Panc 03.27, Capan-1, SW 1990, CFPAC-1, PA-TU-8902
  • the cell lines were obtained from ATCC Depository (Manassas, Virginia), DSMZ (German Collection of Microorganisms and Cell Cultures), JCRB (Japanese Cell Resources Bank) or ECACC (European Collection of Cell Cultures) and cultured in RPMI-1640 media supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine and 10 mM HEPES.
  • the MTT assay (CellTiter 96 ® Non-Radioactive Cell Proliferation Assay from Promega), which is a colorimetric assay based on the ability of viable cells to reduce a soluble yellow tetrazolium salt (MTT) to blue formazan crystals), was used to determine the amount of viable cells after treatment with Apo2L/TRAIL or DR5 antibody.
  • the MTT assay was performed by the addition of a premixed optimized dye solution to culture wells of a 96-well plate containing various concentrations (0 to 1000 ng/ml) of Apo2L/TRAIL or DR5 antibody. During a 4-hour incubation, living cells convert the tetrazolium component of the dye solution into a formazan product.
  • the solubilization/stop solution was then added to the culture wells to solubilize the formazan product, and the absorbance at 570nm was recorded using a 96-well plate reader (SpectraMax).
  • the 570nm absorbance reading is directly proportional to the number of cells normally used in proliferation assays.
  • the absorbance maximum for the formazan product is 570nm and pure solutions appear blue, the color at the end of the assay may not be blue and depends on the quantity of formazan present relative to other components (including serum, acidified phenol red and unreduced MTT) in the culture medium.
  • Cell numbers were optimized by performing a cell titration to produce an assay signal near the high end of the linear range of the assay. Since different cell types have different levels of metabolic activity, this was done for each cell line separately. For most tumor cells examined, 5,000 cells per well to 20,000 cells per well were used.
  • RNA yield Use spectrophotometric analysis to determine the RNA yield. Apply the convention that 1 OD at 260 nm equals 40 ⁇ g/ml RNA.
  • Relative quantitation was performed using the standard curve method. For quantitation normalized to an endogenous control, standard curves were prepared for both the target and the endogenous reference. For each experimental sample, the amount of target and endogenous reference was determined from the appropriate standard curve. Then, the target amount was divided by the endogenous reference amount to obtain a normalized target value. One of the experimental samples served as the calibrator, or 1x sample. Each of the normalized target values was then divided by the calibrator normalized target value to generate the relative expression levels.
  • Figure 5 provides an IC50 summary chart of the data obtained in analyzing non-small cell lung cancer ("NSCLC") cell lines for sensitivity or resistance to apoptotic activity of Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or DR5 monoclonal antibody “mab”, cross-linked “XL” or not crosslinked, + 0.5% fetal bovine serum “FBS” or 10% FBS) as measured in MTT cytotoxicity assays.
  • NSCLC non-small cell lung cancer
  • Figure 6 provides an IC50 summary chart of the data obtained in analyzing pancreatic cancer cell lines for sensitivity or resistance to apoptotic activity of Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or DR5 monoclonal antibody “mab”, cross-linked “XL” or not crosslinked, + 0.5% fetal bovine serum “FBS” or 10% FBS) as measured in MTT cytotoxicity assays.
  • Figures 7 provides an IC50 summary chart of the data obtained in analyzing non-hodgkin's lymphoma cancer ("NHL”) cell lines for sensitivity or resistance to apoptotic activity of Apo2L (+ 10% fetal bovine serum “FBS”) or DR5 monoclonal antibody “mab”, cross-linked “XL” or not crosslinked, + 0.5% fetal bovine serum “FBS” or 10% FBS) as measured in MTT cytotoxicity assays.
  • NDL non-hodgkin's lymphoma cancer
  • Figure 8 provides a comparison of sensitivity (“sen”) or resistance (“RES") of select NSCLC, Pancreatic, and NHL cancer cell lines to DR5 antibody and the correlation to expression of GalNac-T14, as measured by GalNac-T14 mRNA expression.
  • Figure 9 provides a bar diagram graph of various NSCLC, pancreatic, and NHL cell lines ranked (in descending order) by levels of GalNac-T14 mRNA expression patterns.
  • apoptotic cell death program plays important roles in the development and homeostasis of multicellular organisms ( Danial et al., Cell, 116:205 (2004 )). Intracellular stimuli can trigger apoptosis through the cell-intrinsic pathway, which relies on members of the Bcl-2 gene superfamily to activate the apoptotic caspase machinery ( Cory et al., Nat. Rev. Cancer, 2:647 (2002 )).
  • TNF tumor necrosis factor
  • DD functional apoptosis-inducing 'death domain'
  • Fas ligand (FasL) stimulates apoptosis through Fas (Apol/CD95), while Apo-2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) triggers apoptosis through DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2) ( LeBlanc et al., Cell Death Differ., 10:66 (2003 )).
  • DR4 TRAIL-R1
  • TRAIL-R2 DR5
  • FADD Fas associated death domain
  • DISC death-inducing signaling complex
  • Apo2L/TRAIL sensitivity was also examined in vivo using tumor xenografts.
  • tumors derived from the Fut-6-negative colorectal cancer cell lines Colo320 and RKO did not respond to this treatment.
  • siRNAs were synthesized for each gene and verified their ability to reduce target expression by quantitative RT-PCR ( fig. 14A ).
  • siRNA specificity further with a mutant ppGalNacT-14.
  • plasmid containing 6 'silent' nucleotide changes within the siRNA-targeted region ( Editorial, Nat. Cell. Biol., 5:489 (2003 )) ( fig. 14B ).
  • GalNAcT-14 siRNA reduced sensitivity to Apo2L/TRAIL in 4/5 pancreatic cancer and 2/2 melanoma cell lines, while ppGalNAcT-3 or Fut-6 siRNA each reduced sensitivity in 2/3 colorectal cancer cell lines.
  • transfection of PSN-1 or Hs294T cells with GalNAcT-14 siRNA did not alter sensitivity to the topoisomerase II inhibitor etoposide ( fig. 14D ).
  • transfection of PSN-1 or C170 cells with GalNAcT-14 or GalNAcT-3 siRNA did not affect sensitivity to the broad-spectrum protein kinase inhibitor staurosporine ( fig. 14E ).
  • ppGalNAcT-14 Transfection of HEK293 cells with ppGalNAcT-14 revealed cell death when cotransfected with DR4 or DR5, but not the related receptors Fas and TNFR1 or the cell-intrinsic pathway agonist Bax ( Fig. 11D ). Furthermore, ppGalNAcT-14 transfection increased the Apo2L/TRAIL sensitivity of the resistant cell lines H1568 melanoma (Fig. 11E) and PA-TU-8902 and PL-45 pancreatic carcinoma ( Fig. 14F ), but did not alter sensitivity to etoposide (data not shown). In total, GalNAcT-14 overexpression sensitized 4/7 cell lines to Apo2L/TRAIL.
  • Caspase-8 activation requires DISC assembly ( Ashkenazi et al., Science, 281:1305 (1998 )).
  • ppGalNacT-14 nor Fut-6 siRNA substantially altered the amount of DR4 and DR5 in the DISC, or the dose-dependent binding of Apo2L/TRAIL to PSN-1 or DLD-1 cells, which express both DR4 and DR5 ( Fig. 12C , fig. 15B , and data not shown).
  • ppGalNAcT-14 and Fut-6 do not appear to modulate apoptosis by affecting cell-surface receptor levels or Apo2L/TRAIL binding.
  • the extracellular domain (ECD) of human DR5 was expressed in chinese hamster ovary cells, the secreted protein purified, subjected to acid hydrolysis, and the associated monosaccharides were analyzed ( Fig. 13A ). Consistent with the absence of predicted N-glycosylation sites in the DR5 ECD, we did not detect N-linked glycans. However, two samples from 2 independent experiments displayed 3 moles of GalNAc and 3 moles of Gal per mole of DR5 ECD ( Fig. 13A ), suggesting O-linked modification of three sites on DR5 with the core glycan GalNAc-Gal.
  • Protein O-glycosylation modifies serines or threonines.
  • Using a previously established bioinformatics tool for prediction of potential O-glycosylation sites http://wwrw.cbe.dtu.dk/services/NetOGlyc) ( Julenius et al., Glycobiology, 15:153 (2005 )
  • a third site within the alternatively spliced region Fig. 13B ).
  • the first amino acid segment (74-77) contains 3 serines; the second (130-144) has 5 threonines, while the third has 4 threonines and 3 serines.
  • Murine DR5 has sequences similar to the first 2 segments, with 2 serines and 4 threonines, respectively, while human DR4 also has 2 similar sequences containing 1 serine and 5 threonines.
  • DR5 a set of DR5L and DR5S mutants were made, replaced by alanines either the 5 threonines of segment 130-144 (DRSL-5T, DR5S-5T) or these same 5 threonines as well as the 3 serines of segment 74-77 (DR5L-5T3S, DR5S-5T3S).
  • DR5 antibody immunoblot of lysates from HEK293 cells transfected with DR5L or DR5S revealed the presence of the expected DR5L or DR5S bands ( Fig. 13C ).
  • the antibody also detected DR5 bands of higher molecular weight (MW), which became more abundant upon cotransfection of DR5L or DR5S with ppGalNAcT-14 as compared to control ( Fig. 13C , asterisks).
  • MW molecular weight
  • the abundance of these higher MW bands and their augmentation by ppGalNAcT-14 were significantly diminished with DR5L-5T or DR5S-5T and nearly abolished with DR5L-5T3S or DR5S-5T3S, as compared to the wild type constructs.
  • ppGalNAcT-14 mRNA expression below cutoff values (determined at 500 for most cancers and at 200 for skin cancers, Fig. 13E ).
  • the dynamic expression of ppGalNAcT-14 in cancer suggests that this gene, and possibly other related enzymes, may provide useful biomarkers for identifying tumors with greater sensitivity to Apo2L/TRAIL.
  • O-linked glycans display extensive structural diversity, and they modulate various aspects of plasma membrane protein biology, including conformation, aggregation, trafficking, half-life, as well as cell adhesion and signaling activity ( Hang et al., Bioorg. Med. Chem., 13:5021 (2005 ); Hanisch, Biol. Chem., 382:143 (2001 )). Cancer cells often exhibit dramatic alterations in O-glycan profiles, creating unique tumor-associated carbohydrate antigens ( Brockhausen, Biochim. Biophys. Acta, 1473:67 (1999 ); Dube et al., Nat. Rev. Drug Discovery, 4:477 (2005 ); Fuster et al., Nat. Rev. Cancer, 5:526 (2005 )).
  • O-glycosylation also plays an important role in the homing of tumor cells to specific sites of metastasis ( Fuster et al., Cancer Res., 63:2775 (2003 ); Ohyama et al., EMBO J., 18:1516 (1999 ); Takada et al., Cancer Res., 53:354 (1993 )).
  • a significant subset of primary tumor samples from a variety of human cancers shows elevated expression of the O-glycosylation enzyme ppGalNAcT-14, including colon and colorectal cell samples, melanoma cell samples and chondrosarcoma cell samples.
  • Cell culture reagents were purchased from Gibco (Invitrogen/Gibco, Carlsbad, CA), nontagged soluble Apo2L/TRAIL was prepared as described earlier ( Lawrence et al., Nat. Med., 7:383 (2001 )), the O-linked glycosylation inhibitor Benzyl-a-GalNAc was purchased from Calbiochem and all other chemicals (including etoposide and staurosporine) were from Sigma Aldrich (St. Louis, MO).
  • All 119 human carcinoma cell lines were obtained from ATCC or DSMZ (Braunschweig, Germany) and cultured at 37°C and 5% CO 2 in RPMI1640 supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine and 10 mM HEPES without antibiotics like penicillin/streptomycin.
  • 293 human embryonic kidney cells catalog number CRL-1573 were also obtained from ATCC and cultured in 100% Dulbecco's modified Eagle's medium supplemented with 10% FBS.
  • the O-glycosylation mutant CHO cell line, ldlD CHO was licensed from Dr. Monty Kreiger, MIT (Boston MA).
  • IC50 for Apo2L/TRAIL cells were plated in triplicate in 96 well plates, allowed to adhere for 24 hours and then treated with recombinant human Apo2L/TRAIL in increasing concentrations, up to 1000 ng/ml. After a 72h incubation, they were then subjected to a viability assay - MTT assay (Pierce) or CellTiter-Glo Luminescent Cell Viability Assay (Promega) - as per the manufacturer's protocol. Each cell viability experiment was repeated at least three times in low (0.5%) and high (10% FBS) serum and intermediate sensitive cell lines are defined by variability between the IC50s of independent experiments or between low and high serum.
  • a cell line as sensitive based on apoptosis induction of at least 50% of the cells at an Apo2L/TRAIL concentration of 1 ug/ml and as intermediately sensitive based on variability of the amount of apoptosis induced in independent experiments or in presence of low (0.5%) versus high (10%) serum.
  • Apoptosis was quantified by flow cytometric analysis of the average percentage of harvested cells (adherent + floating in the medium) stained with Annexin V (BD Pharmingen).
  • Total cellular RNA was prepared from untreated cells (3 x 10 6 ) using the RNeasy Kit (Quiagen). Labeled cRNA was prepared and hybridized to oligonucleotide microarrays (U133P GeneChip; Affymetrix Incorporated, Santa Clara, CA) as described previously ( Hoffman et al., Nat. Rev. Genetics, 5:229 (2004 ); Yauch et al., Clin. Cancer Res., 11:8686 (2005 )). Scanned image files were analyzed with GENECHIP 3.1 (Affymetrix), Spotfire, GenePattern and Cluster/TreeView.
  • a DNA fragment encoding ppGalNAcT-14 was cloned from cDNA pooled from Apo2L/TRAIL sensitive cell lines and inserted into the expression plasmid pcDNA3.1 (Invitrogen) with an N-terminal Flag tag. This construct was then subjected to site directed mutagenesis (Quikchange Mutagenesis kit, Stratagene) to generate siRNA silent mutants that had 4-6 wobble basepair alterations in the sequence homologous to the siRNA, without changing the protein sequence. The mutations spanned a region of 10bp in the center of the 19bp siRNA binding sequence.
  • DR5Long and DR5Short The DNA sequences for DR5Long and DR5Short, DR4, murine TRAIL receptor, DR4, Fas (variant 1), TNFR1 and Bax (beta variant) were cloned from cDNA pools and inserted into the pRK expression vector (Genentech). O-glycosylation mutants of DR5L and DR5S were generated by site-specific mutation of four threonine to alanine residues, Mut4xTA (T130, T131, T132, T135) or five threonine to alanine residues Mut5xTA (T130, T131, T132, T135, T143).
  • Transient transfection into HEK293 cells with expression constructs of proapoptotic molecules were done in 6 well plates at a concentration of 0.5 ug/well of the proapoptotic molecule and 2.0 ug of ppGalNAcT-14 or a vector control. Cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol. Following a 48h incubation, cells were subjected to apoptosis analysis.
  • ppGalNAcT-14 and mutants were cloned into the pQCXIP retroviral vector (Clontech).
  • High titer retroviral supernatants were generated using the ⁇ NX-Ampho helper cell line.
  • Packaging cells were transfected using Calcium Phosphate (Invitrogen). Supernatants were isolated 48h after transfection and added to target cells along with 10microg/ml polybrene, followed by a 1h centrifugation step at 2700 rpm to enhance infection. Following transduction, cells were subjected to selection with 2microg/ml puromycin.
  • siRNAs against ppGalNAcT-14, ppGalNAcT-3, Caspase-8 and DR5 were designed by Dharmacon (Lafayette, CO) using their proprietary selection criteria. The selected sequences were:
  • Colo205 cells were grown in the presence of Benzyl-a-GalNAc (2mM or 4mM) for 72 hours. At this point they were replated into 96 well plates, allowed to adhere for 24 hours, while still in the presence of the inhibitor. They were then stimulated with increasing concentrations of Apo2L/TRAIL as indicated and subjected to a viability assay.
  • Primer/probe sets for the GalNacT-14 catalog#: Hs00226180_m1_GT14
  • GalNacT-3 catalog#:Hs00237084_m1_GT3
  • GAPDH catalog number of the GAPDH (cat#: 402869) were purchased from Applied Biosystems (Foster City, CA).
  • IP Anti-Apo2L (2E11; ATCC Accession No. HB-12256), anti-DR4 (3G1 and 4G7, ATCC Accession No. PTA-99) and anti-DR5 (3H3, ATCC Accession No. 12534, and 5C7) monoclonal antibodies were generated at Genentech, Inc. using receptor-Fc fusion proteins as antigens.
  • the anti-DR4 (3G1) and anti-DR5 (3H3) monoclonal antibodies used for immunodetection of DR4/5 in DISC immunoprecipitations, were biotinylated using EZ-link Sulfo-NHS-LC biotinulation kit (catalog number 21217) from Pierce.
  • FLAG-tagged Apo2L/TRAIL was prepared and cross-linked with anti-FLAG antibody M2 (Sigma) as described ( Kischkel, Immunity, 12:611 (2000 )).
  • DR4/5 DISC immunoprecipitation experiments also were performed as described, except that anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies were directly conjugated to agarose for the immunoprecipitation ( Sharp et al., J. Biol. Chem., 280:19401 (2005 )).
  • WB 5x10 5 cells per well were seeded in 6 well plates. For RNAi knock-down experiments, cells were treated with different siRNAs for 48h followed by Apo2L/TRAIL for 4, 8 or 24h.
  • Membranes were washed five times with TBS / 0.05% Tween and then incubated with the respective peroxidase conjugated affinity purified secondary antibody (1:5000, Biorad) for 30 min. The membranes were washed again five times and developed using enhanced chemiluminescence (ECL, Amersham) and exposed to Kodak Biomax films.
  • ECL enhanced chemiluminescence
  • TNF family receptors DR4 and DR5 were determined by florescence-activated cell sorting (FACS) using a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA).
  • FACS Fluorescence-activated cell sorting
  • Cells were then washed with PBS and then incubated with a fluorescein (FITC) conjugated goat anti-mouse secondary antibody (Jackson Laboratories) for 30 min at 4°C.
  • FITC fluorescein conjugated goat anti-mouse secondary antibody
  • Caspase-3/-7 activities were assayed at 37°C in 40 ⁇ l of caspase buffer (50mM HEPES pH 7.4, 100 mM NaCl, 10 % sucrose, 1mM EDTA, 0.1% CHAPS and 10 mM DTT) containing 100 ⁇ M of the fluorogenic peptide Ac-DEVD-AFC. Activity was measured continuously over the indicated time by the release of AFC from DEVD-AFC using a Molecular Devices fluorometer in the kinetic mode and with the 405-510 filter pair. For the assessment of caspase activity 20 ⁇ g of total cell protein (Triton X-100 extracts) was used in 40 ⁇ l of caspase buffer (containing 100 ⁇ M DEVD-AFC).
  • Monosaccharide composition of CHO-cell-derived DR5 was obtained after hydrolysis with 4 N TFA. Analysis of the released monosaccharides was carried out on a Dionex BioLC HPLC system using high-performance anion-exchange chromatography coupled to a pulsed amperometric detector.
  • IACAUC Institutional Animal Care and Use Committee

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EP06813477A 2005-08-16 2006-08-15 Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues Active EP1915626B1 (en)

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EP10010134A Withdrawn EP2306200A1 (en) 2005-08-16 2006-08-15 Apoptosis sensitivity to Apo2L/TRAILl by testing for GalNac-T14 expression in cells/tissues
EP06813477A Active EP1915626B1 (en) 2005-08-16 2006-08-15 Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues
EP10010136A Withdrawn EP2287615A1 (en) 2005-08-16 2006-08-15 Apoptosis sensitivity to APO2L/TRAIL by testing for GALNAC-T14 expression in cells/tissues
EP06813452A Active EP1915625B1 (en) 2005-08-16 2006-08-15 Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues

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ZA200800974B (en) 2009-11-25
JP2014237673A (ja) 2014-12-18
US20080182277A1 (en) 2008-07-31
WO2007022214A2 (en) 2007-02-22
ATE540318T1 (de) 2012-01-15
IL188869A0 (en) 2008-04-13
WO2007022157A3 (en) 2007-08-02
HK1115448A1 (en) 2008-11-28
CN101365951A (zh) 2009-02-11
CN101287990A (zh) 2008-10-15
ATE533058T1 (de) 2011-11-15
EP1915625A2 (en) 2008-04-30
ES2380119T3 (es) 2012-05-08
WO2007022214A3 (en) 2007-08-23
KR20080029000A (ko) 2008-04-02
BRPI0615997A2 (pt) 2011-05-31
EP1915626A2 (en) 2008-04-30
JP2011244824A (ja) 2011-12-08
WO2007022157A2 (en) 2007-02-22
IL188874A (en) 2012-10-31
KR100990266B1 (ko) 2010-10-26
ES2376479T3 (es) 2012-03-14
JP2009504186A (ja) 2009-02-05
EP1915625B1 (en) 2012-01-04
US20130072429A1 (en) 2013-03-21
JP5183474B2 (ja) 2013-04-17
AU2010246471A1 (en) 2010-12-16
JP2017129589A (ja) 2017-07-27
AU2010246471B2 (en) 2011-06-23
CA2619759A1 (en) 2007-02-22
US20090004204A1 (en) 2009-01-01
RU2409817C2 (ru) 2011-01-20
ZA200800969B (en) 2009-08-26
NO20081374L (no) 2008-05-16
EP2287615A1 (en) 2011-02-23
HK1115638A1 (en) 2008-12-05
RU2010144511A (ru) 2012-05-10
JP5186372B2 (ja) 2013-04-17
RU2008110089A (ru) 2009-09-27
RU2008110078A (ru) 2009-09-27
IL188874A0 (en) 2008-04-13
RU2416097C2 (ru) 2011-04-10
JP2014237672A (ja) 2014-12-18
KR100990273B1 (ko) 2010-10-26
IL188869A (en) 2012-10-31
DK1915626T3 (da) 2012-02-13
CA2619842A1 (en) 2007-02-22
CN101287990B (zh) 2012-07-04
PT1915626E (pt) 2012-02-03
NO20081375L (no) 2008-05-16
CN101365951B (zh) 2013-09-11
AU2006279578A1 (en) 2007-02-22
EP2306200A1 (en) 2011-04-06
BRPI0616005A2 (pt) 2011-05-31
AU2006279618A1 (en) 2007-02-22
AU2010246471B9 (en) 2012-08-02
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